Nanoscale pattern of steric hindrance in PEG copolymers improve potency of patient-derived human mesenchymal stem cells
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1
Vanderbilt University, Biomedical Engineering, United States
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2
Imperial College London, Materials, United Kingdom
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3
Vanderbilt University, Cell and Developmental Biology, United States
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4
Rutgers University, Biomedical Engineering, United States
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5
Vanderbilt University, Medicine, United States
Introduction: One bottleneck in translation of stem cell therapies is the inability to create sufficient cell mass via in vitro expansion, which demonstrate passage-associated abnormalities (e.g. human mesenchymal stem cell: hMSCs). Molecular scale analyses of how alterations in substrate chemistry changes stem cell behavior at the cell-material interface have not been reported clearly in the literature. Without a mechanistic understanding of how cells sense and respond to nanoscale changes of synthetic substrates, the design of cell-instructive scaffolds that harness the therapeutic potential of hMSCs cannot be completed.
Materials and Methods: Diblock poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) polymers with varying (1) PEG chain sizes (750 Da, 2 kDa, and 5 kDa) and (2) molar compositions (5-20% PEG) were used. Commercial hMSCs from Lonza were cultured on spin-coated copolymers for the entire library to characterize cell attachment, and 4 copolymers (PCL-b-5.5%PEG750, PCL-b-20.6%PEG750, PCL-b-5.6%PEG2k and PCL-b-9.3%PEG2k) were further tested for cell phenotype characterization by qPCR, western blot, immunofluorescent imaging and flow cytometry. X-ray scattering was performed to elucidate the packing structure of the PCL-PEG films. Structured illumination imaging visualized cell attachment machinery to correlate images with the x-ray scattering data. Finally, patient-derived hMSCs from male donors over 65-years old were cultured on the select copolymers to verify the same cellular response in a broader donor population.
Results and Discussion: 750 Da PEG copolymers allowed for cell attachment while samples with over 9.3 mole% for 2kDa PEG had reduced or inhibited cell attachment, and no cell attachment was observed for 5 kDa PEG copolymers. hMSCs showed modest aggregation on 750 Da copolymers, but 2 kDa PEG copolymers promoted distinct hMSC spheroid-like aggregates. The gene expression of SOX2 and NANOG was significantly upregulated relative to TCPS on all select copolymers. Both proliferation rate and ROS load of hMSCs (senescence markers) were significantly decreased when cultured copolymers. The x-ray scattering data indicate that 1) the PEG segments are not located in crystalline PCL areas but 2) form phase-separated domains in the amorphous PCL area; and 3) 2 kDa and 5 kDa PEG are excluded from the crystalline PCL lamellae with formation of a mushroom-like structure while 750 Da PEG stayed embedded in the amorphous PCL. Structured illumination microscopy of paxillin, a mature focal adhesion marker, verified that hMSC attachment was influenced by exposure to limited amorphous PCL, and that this cell-biomaterial phenomenon was responsible for altering stem cell potency as the 2k PEG copolymers had drastically increased focal adhesion quantity and striated morphology than that of 750 Da PEG copolymers, 100%PCL and glass substrates. hMSCs from patients over 65-years old also displayed the same phenotype response.
Conclusion: The variations of the PEG content and chain length in copolymers provides new insight into the role of polymer chemical composition and the resulting nanoscale structures in improving the therapeutic potential of hMSCs from both commercial and patient sources. These data show promising translation potential of copolymers as a new class of culture materials for adult stem cell therapy.
Keywords:
stem cell,
Polymeric material,
bioinerface,
matrix-cell interaction
Conference:
10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.
Presentation Type:
New Frontier Oral
Topic:
Interfacial phenomena
Citation:
Balikov
D,
Crowder
SW,
Lee
J,
Gupta
MG,
Fenix
AM,
Burnette
DT,
Murthy
S and
Sung
H
(2016). Nanoscale pattern of steric hindrance in PEG copolymers improve potency of patient-derived human mesenchymal stem cells.
Front. Bioeng. Biotechnol.
Conference Abstract:
10th World Biomaterials Congress.
doi: 10.3389/conf.FBIOE.2016.01.02090
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.
*
Correspondence:
Dr. Daniel Balikov, Vanderbilt University, Biomedical Engineering, Nashville, TN, United States, Email1
Dr. Spencer W Crowder, Imperial College London, Materials, London, United Kingdom, spencer.crowder@gmail.com
Dr. Jung Bok Lee, Vanderbilt University, Biomedical Engineering, Nashville, TN, United States, jung.bok.lee@vanderbilt.edu
Dr. Mukesh G Gupta, Vanderbilt University, Biomedical Engineering, Nashville, TN, United States, mukesh.k.gupta@Vanderbilt.Edu
Dr. Aidan M Fenix, Vanderbilt University, Cell and Developmental Biology, Nashville, TN, United States, aidan.m.fenix@vanderbilt.edu
Dr. Dylan T Burnette, Vanderbilt University, Cell and Developmental Biology, Nashville, TN, United States, dylan.burnette@vanderbilt.edu
Dr. Sanjeeva Murthy, Rutgers University, Biomedical Engineering, Piscataway, NJ, United States, Murthy@dls.rutgers.edu
Dr. Hak-Joon Sung, Vanderbilt University, Biomedical Engineering, Nashville, TN, United States, hak-joon.sung@vanderbilt.edu